CN117701762A - Molecular marker related to seed setting rate of broccoli and application thereof - Google Patents
Molecular marker related to seed setting rate of broccoli and application thereof Download PDFInfo
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Abstract
The invention discloses a molecular marker related to seed setting rate of broccoli and application thereof, wherein the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the 51 st position of the sequence has C/T polymorphism, the SNP is positioned at 37728588 th position of C6 chromosome of a broccoli HDEM (verson 1.0) reference genome, and when the base sequence is mutated from C to T, the seed setting rate of the corresponding broccoli is improved. Screening the broccoli germplasm with high setting rate by utilizing SNP molecular markers, carrying out PCR amplification by using KASP primers, exciting HEX fluorescence by the primers in the broccoli material with low setting rate, and exciting FAM fluorescence by the SNP in the broccoli material with high setting rate, thereby realizing early and rapid identification of broccoli germplasm resources by utilizing molecular biological means.
Description
Technical Field
The invention relates to a molecular marker related to a setting percentage, in particular to a molecular marker related to a setting percentage of broccoli seeds and application thereof.
Background
The broccoli (Brassica oleracea. Var. Itica) is rich in nutrients such as glucosinolates, and the relative lag of seed production technology and serious deficiency of seed production yield are important problems faced by the broccoli industry.
Currently, related studies on the seed setting rate of crops have focused mainly on model plant rice, related genes such as POLLEN TUBEBLOCKED (PTB 1) being able to positively regulate the seed setting rate of rice ears by promoting pollen tube growth (Li S, li W, huang B, et al Natural variation in PTB1regulates rice seed setting rate by controlling pollen tube growth. Nat Commun.2013;4 (1): 2793.); the OsCNGC13 gene can change Ca in stigma 2+ Is used to promote double fertilization and fruiting in rice (Xu Y, yang J, wang Y, et al, osCNGC13 proteins seed-setting rate by facilitating pollen tube growth in stylar tissues, paltive R, ed. PLOS Genet.2017;13 (7): e 1006906). It has recently been found that mutation of a novel gene LOWSEED SETTINGRATE (LSSR 1) in rice can cause abnormal pollen grain germination, thereby affecting the rice setting rate (Xiang X, zhang P, yu P, et al LSSR1 facilitates seed setting rate by promoting fertilization in rice. Rice.2019;12 (1): 31). In brassica crops, the phenomenon of "self-incompatibility" is considered to be an important factor affecting seed setting rate. Numerous studies have shown that brassica crops such as broccoli belong to the typical sporophyte self-incompatibility type, genetically controlled by 1S-locus with multiple alleles, also known as S haplotypes. Only male determinants (SCR, serine/threonine receptor kinase) of male parent and female determinants (SRK, cysteine-rich protein) of female parent belong to different S haplotypes, pollen falling on the column head can germinate and grow, and fertilization is completed to produce seeds. In addition to the S site, many other genes are involved in the self-incompatibility reaction, including genes such as MLPK (M-site protein kinase), ARC1 (brachytherapy protein), THL1/2 (thioredoxin h), exo70A1 (exocytosis complex), GLO1 (glyoxalase), and the like. These genes act upon recognition of SCR by SRK, forming a signaling pathway, ultimately leading to self-incompatibility. Recent researches have found that pollen in crops such as cabbage can activate through stigma SRK receptorThe downstream FERONIA receptor kinase signaling pathway elevates active oxygen in the stigma, thereby inhibiting the entry of exogenous pollen into the pollen tube (Huang J, yang L, yang L, et al Stigma receptors control intraspecies and interspecies barriers in. Brassica cae. Nature.2023;614 (7947): 303-308). We have found that the major domestic broccoli variety and breeding material contain 19S haplotypes, and more importantly that some breeding parents exhibit a phenomenon of very low fruiting rate even though they contain different S haplotypes (Yu HF, zhao ZQ, sheng XG, et al identification of S-bioplotypes in DH-lines of broccoli (Brassica oleracea L. Var. Identification). J Hortic Sci Biotechnol.2014;89 (4): 430-434), suggesting that this trait may also be controlled by other unknown genetic loci and regulatory networks.
Therefore, the gene locus related to the seed setting rate in the broccoli is explored, and the molecular marker closely linked with the gene is developed, so that the seed setting rate of the broccoli can be identified efficiently and accurately, the seed yield of the broccoli is improved, and an important technical support is provided for guaranteeing the localization process of the broccoli variety.
Disclosure of Invention
The invention aims to provide a molecular marker related to the seed setting rate of broccoli and application thereof, and the molecular marker is used for screening broccoli germplasm with high setting rate, so that early and rapid identification of broccoli germplasm resources is realized by using a molecular biological means.
In order to achieve the above purpose, the invention provides a molecular marker related to the seed setting rate of broccoli, and the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the 51 st position of the sequence has C/T polymorphism, and when the base sequence is mutated from C to T, the seed setting rate of the corresponding broccoli seeds is improved.
The invention also aims to provide the molecular marked primer group, and the nucleotide sequence of the primer group is shown as SEQ ID NO. 2-4.
Preferably, the 5' end of the primer with the nucleotide sequence shown as SEQ ID NO.2 is added with a universal joint sequence with the nucleotide sequence shown as SEQ ID NO. 5; the 5' end of the primer with the nucleotide sequence shown as SEQ ID NO.3 is added with a universal joint sequence with the nucleotide sequence shown as SEQ ID NO. 6.
It is another object of the present invention to provide a KASP kit related to the seed setting rate of broccoli, wherein the primer set in the KASP kit is the primer set.
The invention also aims to provide application of the molecular marker or the primer group in detecting the genotype of broccoli, screening high seed setting rate or molecular marker assisted selective breeding.
It is another object of the present invention to provide a method for identifying the seed setting rate of broccoli, the method comprising:
(1) Extracting genome DNA of the broccoli to be detected;
(2) Performing PCR amplification using the primer set according to claim 2 or 3;
(3) Analyzing the PCR amplification product by adopting a fluorescence detector, and judging the seed setting rate of the broccoli according to the genotyping result: if the genotyping result is CC, the method is a broccoli with low fruiting rate; if the genotyping result is TT, the method is a high-setting-rate broccoli; if the genotyping result is CT, the genotyping result is between the low-setting-rate broccoli and the high-setting-rate broccoli.
Preferably, the primer excites HEX fluorescence in the low setting rate broccoli material and excites FAM fluorescence in the high setting rate broccoli material.
Preferably, the PCR reaction procedure is: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 30s, annealing at 60-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 95℃for 30s and annealing at 55℃for 60s for 26 cycles.
Preferably, the PCR reaction system comprises: a PCR premix, a mixture of the primer set according to claim 2 or 3, and a template DNA.
The molecular marker related to the seed setting rate of broccoli and the application thereof have the following advantages:
the invention constructs a high-density genetic linkage map containing 4425 markers, positions the gene for controlling seed setting rate in the region of 698.9-725.4cM of C6 chromosome, the LOD value of the QTL is 36.2, the interpretable phenotype variation is 59.8%, and the reliability of the locus is verified, so that the co-positioned QTL has stable regulation and control effect on setting rate of broccoli seeds under different genetic backgrounds.
The invention develops a SNP marker KM268 closely linked with the seed setting rate of broccoli, wherein the SNP is positioned at the base C or T at 37728588 position of a C6 chromosome of a broccoli HDEM (verson 1.0) reference genome, when the base sequence is mutated from C to T, the seed setting rate of the corresponding broccoli is obviously improved, thereby realizing early and rapid identification of broccoli germplasm resources by utilizing a molecular biological means.
Drawings
FIG. 1 is a phenotype and genetic map for the localization of a set rate QTL; wherein A is F2 colony constructed by hybridization of 3 parent material seed setting rate phenotypes utilized by the invention; b is a BJ-F2 population individual seed setting rate phenotype distribution histogram; c is the distribution of SNP markers of BJ-F2 population on chromosome; the color represents the density of marks within a 10cM interval.
FIG. 2 shows the results of the QTL positioning of the seed setting rate characteristics of BJ-F2 populations.
FIG. 3 shows the result of the QTL positioning of the seed setting rate property of the BC-F2 population.
FIG. 4 is a gene distribution scatter plot of GSL-OH gene SNP primers based on KASP technology; wherein, the abscissa is FAM fluorescence value, and the ordinate is HEX fluorescence value.
FIG. 5 is a box plot of the phenotype distribution of marker KM268 in the F2 population, closely linked to the seed setting rate of broccoli.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Experimental example 1 development of molecular markers related to seed setting Rate based on QTL positioning of different genetic populations
1. Positioning of QTL (quantitative trait locus) of seed setting rate of broccoli
The invention utilizes the broccoli materials B58-6 and J1402 with obvious difference of self-maturing rate to hybridize, thus constructing BJ-F containing 214 strains 2 Population (FIG. 1A). BJ-F was obtained based on the number of seed set per individual plant 2 The seed setting rate of the population was divided into five classes (0, 1, 2, 3, 4), and the population phenotype was found to exhibit a normal distribution (B of fig. 1), and the seed setting rate of the individual plants was expressed as 1:3 (χ) 2 ＝0.22，P>0.05 Indicating that the setting rate of the population is controlled by a single gene. The invention utilizes a pre-developed broccoli 40K SNP liquid phase chip to genotype the population, and constructs a high-density genetic linkage map containing 4425 markers (see figure 1). Using WinQTL2.5 software, the gene controlling seed setting rate was located within the region 698.9-725.4cM of the C6 chromosome (see FIG. 2), the LOD value of this QTL was 36.2 and the explained phenotypic variation was 59.8%.
To verify the reliability of this site, the broccoli farmer species BR06 and C4101 were hybridized to construct BC-F containing 212 lines 2 The population, by genetic analysis, also mapped to a major QTL on the C6 chromosome (see fig. 3), interpreted a phenotypic variation of 23.7%. BJ-F 2 And BC-F 2 The located QTL related to the seed setting rate is compared with the HDEM reference genome, and the physical positions of the three QTLs are overlapped, which indicates that the co-located QTL plays a stable role in regulating and controlling the seed setting rate of broccoli seeds under different genetic backgrounds.
2. Development of KASP markers related to seed setting rate of broccoli in QTL interval
By using the QTL interval obtained above, the genetic position of the interval is compared with the HDEM (verson 1.0) reference genome to obtain the physical position of the interval. According to the result of the parent re-sequencing, SNP loci with genotypes different in the physical interval are selected and developed into KASP markers. Through further screening and verifying genetic groups, the KASP marker KM268 which has good genotyping result and is closely related to the seed setting rate of broccoli is used as a seed setting rate molecular marker of broccoli for rapid and accurate surface type screening. The SNP is located at base C or T at position 37728588 of the C6 chromosome of the broccoli HDEM (verson 1.0) reference genome (see FIG. 4). When the base sequence is mutated from C to T, the seed setting rate of the corresponding broccoli seeds is obviously improved.
KASP marker KM268 (SEQ ID No. 1), specifically as follows:
TTGTCAGCCTGATCATTCACCTCACCGTTGTCAGAAACAGACATCT CCTTCTGAGAAATCTCAACGCACAAGTCTTTCGTCCAAGAGGTAACAA TTTCCAT。
the KM268 primer is specifically as follows:
the nucleotide sequence of forward primer FAM (SEQ ID NO. 2) is:
GTGCGTTGAGATTTCTCAA；
the nucleotide sequence of the forward primer HEX (SEQ ID NO. 3) is:
GTGCGTTGAGATTTCTCAG；
reverse primer (SEQ ID NO. 4):
CAAGGACATTCCAGCTAGACAA。
the 5' ends of the 2 upstream primers are respectively added with a universal joint sequence, and the specific steps are as follows:
the universal linker sequence added to forward primer FAM (SEQ ID No. 5) is:
GAAGGTGACCAAGTTCATGCT；
the universal linker sequence added to forward primer HEX (SEQ ID NO. 6) is:
GAAGGTCGGAGTCAACGGATT。
the two forward primers in the primer pair have base difference at the 3' -end, can be competitively combined with a target site, display corresponding FAM or HEX fluorescence, and can judge the genotype of the target site after signal amplification.
Experimental example 2 verification of broccoli seed setting Rate-related marker KM268 in isolated populations
1. Extraction and purification of DNA of isolated population to be detected
And screening the individuals subjected to chromosome recombination in the QTL interval from BJ-F2 groups consisting of 214 individuals to carry out selfing, so as to construct BJ-F2:3 separation groups. 150 single plants are randomly selected, blade DNA is extracted by using a CTAB method, and the quality and concentration of the DNA are detected by using a spectrophotometer.
2. Genotyping of isolated populations using KM268
PCR amplification was performed by adding specific KASP Primermix (KASP primer mix) and universal KASP Mastermix to the extracted DNA template. Wherein, the KASP Master mix comprises the following components: universal FRET cassette fluorescent primer, ROX reference dye, klearTaq DNA polymerase, dNTP and MgCl 2 The method comprises the steps of carrying out a first treatment on the surface of the The PCR reaction system for KASP detection is as follows: PCR premix 5. Mu. L, KASP primer mix 0.14. Mu.L (where the final concentration of each primer is 5 nM) and 20 ng/. Mu.L template DNA 5. Mu.L; the reaction conditions for PCR were: pre-denaturation at 94℃for 15min; denaturation at 94 ℃ for 20s, annealing at 61-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 94℃for 20s and annealing at 55℃for 60s for 26 cycles;
as a result, as shown in Table 1, of 150 individuals, 30 strains had genotype of CC, 48 strains had genotype of TT, and 72 strains had genotype of CT.
TABLE 1
3. Measuring seed setting rate of segregating population
Seed setting numbers of each 20 siliques in 150 individual plants were measured, and seed setting rates (seed setting rate=total number of seeds/siliques) of each individual plant were counted, and seed setting rates thereof were from 0 to 17, as shown in table 1.
Lines of different genotypes of KM268 were counted and a box graph was constructed as shown in FIG. 5. The average seed setting rate of CC genotype single plant is 0.18, while the average seed setting rate of TT genotype single plant is 10.91, t-test shows that there is very obvious difference (P < 0.001) between the phenotypes of two different genotypes, which shows that the marker can be used to distinguish the setting rate of broccoli seed effectively and accurately.
While the present invention has been described in detail through the foregoing description of the preferred embodiment, it should be understood that the foregoing description is not to be considered as limiting the invention. Many modifications and substitutions of the present invention will become apparent to those of ordinary skill in the art upon reading the foregoing. Accordingly, the scope of the invention should be limited only by the attached claims.
Claims (9)
1. The molecular marker related to the seed setting rate of broccoli is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 1; the 51 st position of the sequence has C/T polymorphism, and when the base sequence is mutated from C to T, the seed setting rate of the corresponding broccoli seeds is improved.
2. The molecular marker primer set according to claim 1, wherein the nucleotide sequence of the primer set is shown in SEQ ID NO. 2-4.
3. The primer group according to claim 2, wherein the 5' -end of the primer having the nucleotide sequence shown in SEQ ID NO.2 is added with a universal adaptor sequence having the nucleotide sequence shown in SEQ ID NO. 5; the 5' end of the primer with the nucleotide sequence shown as SEQ ID NO.3 is added with a universal joint sequence with the nucleotide sequence shown as SEQ ID NO. 6.
4. A KASP kit related to the seed setting rate of broccoli, wherein the primer set in the KASP kit is the primer set according to claim 2 or 3.
5. Use of the molecular marker of claim 1 or the primer set of claim 2 or 3 for detecting broccoli genotype, screening for high seed setting rate or molecular marker assisted selection breeding.
6. A method of identifying a seed setting rate of broccoli, the method comprising:
(1) Extracting genome DNA of the broccoli to be detected;
(2) Performing PCR amplification using the primer set according to claim 2 or 3;
(3) Analyzing the PCR amplification product by adopting a fluorescence detector, and judging the seed setting rate of the broccoli according to the genotyping result:
if the genotyping result is CC, the method is a broccoli with low fruiting rate;
if the genotyping result is TT, the method is a high-setting-rate broccoli;
if the genotyping result is CT, the genotyping result is between the low-setting-rate broccoli and the high-setting-rate broccoli.
7. The method of claim 6, wherein the primer excites HEX fluorescence in low-setting broccoli material and excites FAM fluorescence in high-setting broccoli material.
8. The method of claim 6, wherein the PCR reaction procedure is: pre-denaturation at 95℃for 10min; denaturation at 95 ℃ for 30s, annealing at 60-55 ℃ for 60s, and annealing temperature of each cycle is reduced by 0.6 ℃ for 10 cycles; denaturation at 95℃for 30s and annealing at 55℃for 60s for 26 cycles.
9. The method of claim 6, wherein the PCR reaction system comprises: a PCR premix, a mixture of the primer set according to claim 2 or 3, and a template DNA.
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| CN202311854669.2A Pending CN117701762A (en) | 2023-12-29 | 2023-12-29 | Molecular marker related to seed setting rate of broccoli and application thereof |
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| Country | Link |
|---|---|
| CN (1) | CN117701762A (en) |
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2023
- 2023-12-29 CN CN202311854669.2A patent/CN117701762A/en active Pending
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